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Project summary The dense-core vesicle is an organelle that releases peptide hormones, growth factors, and biogenic amines from neurons and neuroendocrine cells in response to increases in calcium. Dense-core vesicle cargos regulate a variety of biological processes including neuronal survival and development, pain sensation, blood glucose homeostasis, and synaptic plasticity. As a consequence, numerous diseases such as mood disorders, obesity, and diabetes are caused by defects in neuropeptide and hormone secretion. Thus, it is important to determine the molecular machinery and mechanisms that orchestrate the biogenesis and release of these vesicular carriers. However, in comparison to other vesicular compartments such as synaptic vesicles, little is known about the molecular mechanisms of dense-core vesicle biogenesis, trafficking, and release. Dense-core vesicles are generated at the trans-Golgi and gain their compartmental identity in a poorly defined maturation process that occurs post-Golgi, but few molecules have been identified that function in these processes. Cargo sorting to dense-core vesicles remains a puzzle. The long-term goal of this project is to understand the molecular mechanisms by which dense-core vesicles are formed, sort cargos, and gain their compartmental identity. A first step towards this goal is to identify molecules required for dense-core vesicle biogenesis. We performed a genetic screen in the nematode C. elegans for mutants defective in dense-core vesicle function and identified a number of new proteins that act in dense-core vesicle biogenesis, including the small GTPase RAB-2 and CCCP-1, a RAB-2 effector, as well as the EARP endosomal trafficking complex. In this project, we will test how RAB-2 and its effectors interact with EARP to mediate cargo sorting to dense-core vesicles. In Aims 1 and 2, we will use genetic, biochemical and cell biological approaches in C. elegans and in the mammalian insulin-secreting cell line INS-1 832/13 to determine how and where the RAB-2 and EARP complexes are localized, whether they physically and functionally interact, and determine their precise roles in the sorting, processing, and secretion of dense-core vesicle cargos. In Aim 3, we will use a combination of in vitro biochemical approaches and in vivo functional assays to determine how the golgin-like coiled-coil protein CCCP-1 binds membranes and functions to mediate dense-core vesicle biogenesis. These studies will advance our understanding of how dense-core vesicles are generated and sort cargos, and provide general insights into mechanisms of membrane trafficking and cargo sorting controlled by multisubunit complexes that mediate trafficking between endosomal compartments and the trans-Golgi network.